Please mention what protein you are looking for..because, if it is a phospho protein, blocking should NOT be done in milk.

Try this:

1. Blocking should be done before primary antibody treatment, 1 hour at room temp.

2. Use 3%BSA in TBST (0.1% tween) for blocking instead of skimmed milk.

3. DO NOT wash with TBST after blocking.

4. Do the primary antibody dilution also in 3% BSA in TBST.

5. Use Tris buffered saline with 0.1% tween (0.1%TBST) instead of PBST for washing.

6. wash minimum three times in TBST for five minutes every time after primary and secondary treatment.

7. MOST IMPORTANTLY, during the whole procedure NEVER LET THE BLOT DRY..! always let it stay in TBST.

Hope this is helpful. please revert for any further clarifications.

for catin 42 KDA protien as loading control,sad am notgetting band

Actin

The background is oftenly due to insuffcient blocking, insuffcinet washing or some times due to primary antibody. 1h blocking with skimmed milk is suffcient enough. Also incubation with primary antibody for 2h at RT or 4C overnight is OK, but sometimes we get background because of the problem in the antibody. We do memeberane washing three times each for 10-15 min. Also your detection procedure where you mix luminol, cumaric acid and H2O2 might have some problem. In our lab we use ECL mxiture from the company, working perefectly good.

Actin, tubulin are abundantly expressed and very easy to be detected, just few seconds exposure gives a very strong and clear band......

I am little skeptical about your protocol. Blocking is done after the wet or semi-dry western blot step and before primary Ab incubation. Otherwise, high background is usually due to

1. too much HRP in your system (HRP conjugated Ab).

2. Try and see using protein free blocking buffer.

3. Dilute your detection reagent, ECL with H2O and start with highly diluted ECL solution.

Good Luck!

Please mention what protein you are looking for..because, if it is a phospho protein, blocking should NOT be done in milk.

Try this:

1. Blocking should be done before primary antibody treatment, 1 hour at room temp.

2. Use 3%BSA in TBST (0.1% tween) for blocking instead of skimmed milk.

3. DO NOT wash with TBST after blocking.

4. Do the primary antibody dilution also in 3% BSA in TBST.

5. Use Tris buffered saline with 0.1% tween (0.1%TBST) instead of PBST for washing.

6. wash minimum three times in TBST for five minutes every time after primary and secondary treatment.

7. MOST IMPORTANTLY, during the whole procedure NEVER LET THE BLOT DRY..! always let it stay in TBST.

Hope this is helpful. please revert for any further clarifications.

I think you should do blocking for more time followed by sufficient washing after removing the primary and secondary antibody with TBST. You havent mentioned the duration of your washing time. And as mentioned in the above comments, actin is a well expressed protein so you will get proper band for it. Are you using cell lines or tissue for the western blot and what is the dilution of the antibody you were using. ???

Thank you all for suggestion,yes blocking i do before pr ab for 1 hr in skimmed milk,i took fresh milk and increased upto 1 and half hr,it worked.no background and did proper washing,increased 1 time.yes actin is expressed so abundantly and it was sad.plus i am doing western first time.my ecl sloution ,luminol-cumaric caid forms complex then,addition of hydrogen peroxide in tris diluted one ph-8 ,there i was hurrying up.i easily added 50 to 100 ul of ecl sol for two bands and waited for the rxn and i could easily see the signal.

Thanks a lot and i am seeing protein expression in mcf-7 breast cancer cell lines.

Rest bsa is expensive,and cant use.

Thanks a lot

BSA is cheap if you buy it from Santa Cruz, It is working very good in blocking of westerns